Prepareration Of Monoclone Antibody And Establishment Of Elisa Rapid Detecting Method Of Sulfaquinoxaline

Sulfaquinoxaline (Sulfaquinoxaline, SQ) is a highly effective anticoccidial agent and antibacterial agents, mainly used for treating the coccidiosis of poultry, young ruminant animals,and small animals. At farms SQ is added to animal feed or drinking water in the process of feeding to combat animal diseases, thus chronic poisoning of livestock and poultry and drug residues in poultry products are easily occured. If Sulfa-quinoxaline residues which containing in the animal food was intaked by human, and up to a certain concentration, will cause people's abnormal reaction, allergic reactions, Distortion, cancer and other toxic side effects. China and other Western developed countries like the European Union, Japan, and the United States all have arequired standard that Sulfaquinoxaline's residue must be less than 10~0ng/g. At present, immunological analysis method has become a hot research in the field of drug residues detected for its advantages of convenient, rapid, high sensitivity and specificity, low cost. This study was designed to establish enzyme-linked method to detecting Sulfaquinoxaline residual, thus would facilitate the livestock farmers'self-test, and provide a better studies basis.for effectively monitor the using and residual of Sulfa-quinoxaline.This study synthesized the original immunogen BSA-SQ by diazotization after analysising on the structure of Sulfaquinoxaline and its carrier protein(BSA, OVA), and then prepared SQ monoclonal antibody, developed the ultimate success of the directly competitive ELISA rapid detecting Kit for Sulfaquinoxaline. Key findings are as follows:1. The preparation and identification of Sulfaquinoxaline's complete antigen Respectively, using glutaraldehyde (GA), carbodiimide (EDC), diazotization to explore artificial antigen BSA-SQ's coupling preparation, and initially identificated their coupling effects using UV scanning (UV), SDS-PAGE electrophoresis. These results showed that: The results of using GA and EDC couplled BSA and SQ were not satisfactory, Preparation of BSA-SQ by the diazotization is effectively.Used the artificial antigen BSA-SQ prepared by the diazotization to immune BALB/C mice, received a high titer anti-SQ polyclonal antibody (SQ pAb), this confirmed the successful preparation of artificial antigen BSA-SQ by diazotization.2. The preparation and immunological characteristics's identification of Sulfa-quinoxaline monoclonal antibodies BABL/C mice produced the polyclonal antibody after were immuned the BSA-SQ, then they were screened by ELISA test for used as a spare cell mouse; this mouse was ultra-freed, then its tumor cells and the NS0 spleen cells were treated with PEG-4000 for cell fusion, these fused cells were selectly cultured by HAT medium;then after using limited dilution cloning for three times.I gained three hybridoma cell strain producting anti-SQ monoclonal antibody(SQ mAb): 1H8-B11, 1H8-C6, 1H8-F5; and used the method of inducing ascites in vivo gaining Large-scale production and purificated it. After identified, The indirect ELISA titers of 1H8-B11, 1H8-C6, 1H8-F5 cell culture supernatant were 1:1.92×10~2,1:1.92×10~2,1:3.84×10~2, ascites titer of them were 1:2.0×10~5,1:2.0×10~5,1:2.56×10~5. The affinity tests of them showed that affinity constant (Ka) of cell line 1H8-F5 was the highest,which is 4.19×10~9 L/moL. Blocking ELISA measuring SQ IC50 of cell line 1H8-F5 was 13.1 ng/mL, and cell line 1H8-F5 has a cross-reactivity rate of 20% to the pyrazine sulfonamide sodium chloride, has not CR with other similar compounds.3. The establishment of enzyme-linked immunosorbent rapid detection method for SulfaquinoxalineOn the basisness of having obtained the SQ mAb and the principle of direct competition ELISA experiment, SQ rapid detection of residues in direct competition ELISA Kit (SQ-Kit) was assembly prepared. SQ-Kit standard's curve regression equa-tion was y =- 0.262x +0.6778, correlation coefficient is R~2 = 0.9786, its SQ IC50 is 7.5ng /mL, has a detection limit of 5.0ng/mL and a Linear detection range of 5.0 ~ 128.0ng/mL, it also has advantages of good specificity, reproducibility and stability. The work for SQ-Kit detecting the Sulfaquinoxaline remaining in the milk and meat samples can be completed within the 1.5 ~ 2h.The SQ-Kit has a fast, simple, sensitive, specific characteristics, is suitable for the ladge scaled rapid qualitative and quantitative identification detection of SQ residues.